# Multivalent Tranexamic Acid (TXA) and Benzamidine Derivatives for Serine Protease Inhibition

## TL;DR

Scientists designed new drug-like molecules that better block blood-clotting enzymes by combining two inhibitors with different linker lengths.

## Contribution

New multivalent inhibitors with dPEG linkers showed improved serine protease inhibition through multivalent subsite binding effects.

## Key findings

- Heterobivalent molecules with dPEG linkers improved inhibition of plasmin and tPA.
- Homomultivalent TXA (PAMAM8-TXA) strongly inhibited plasmin with a Ki of 2.5 ± 1.8 μM.
- IC50 values were measured using fluorescent fibrin clots to assess kringle binding effects.

## Abstract

Blood coagulation
and fibrinolysis pathways involve many serine
proteases in a careful equilibrium. Disruption of this hemostatic
balance can cause life-threatening thromboembolic and bleeding disorders
that require therapeutic intervention. Heterobivalent molecules synthesized
with both benzamidine (active site serine protease inhibitor) and
tranexamic acid (TXA, kringle/lysine-site inhibitor) of increasing
dPEG linker lengths (dPEG4–dPEG36) were
synthesized and analyzed for plasmin, thrombin, and tissue plasminogen
activator (tPA) inhibition using soluble enzymatic substrates. Linker
lengths greater than the active and lysine binding site separation
achieved improved inhibition with plasmin and tPA due to multivalent
subsite binding effects. Despite TXA being a weak active site inhibitor,
homomultivalent TXA (PAMAM8-TXA) demonstrated strong competitive
plasmin inhibition (K
i = 2.5 ± 1.8
μM) due to the statistical rebinding effect. IC50 values were also determined by assaying on physiologically relevant,
fluorescently tagged, annular fibrin clots to capture the effect of
kringle binding inhibition on fibrinolytic potential in the presence
and absence of inhibitors.